Ten Cases of Actinobaculum schaalii Infection: Clinical Relevance, Bacterial Identification, and Antibiotic Susceptibility
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微生物临床杂志 2005年第10期
Department of Clinical Microbiology, Viborg Hospital, Viborg
Department of Clinical Microbiology, Odense Hospital, Odense
Unit of Clinical Microbiology, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen
Departments of Medicine
Surgery, Roskilde Hospital, Roskilde, Denmark
ABSTRACT
Nine of 10 strains of Actinobaculum schaalii caused urinary tract infections in predisposed individuals. Identification included 16S rRNA gene sequence analysis and use of the API Coryne and Rapid ID32A test systems. A. schaalii is easily overlooked due to its slow growth in ambient air and its resemblance to the normal bacterial flora on skin and mucosa.
TEXT
The genus Actinobaculum, first described in 1997, includes the Actinobaculum suis and Actinobaculum schaalii species. A. suis is an important cause of urinary tract infections (UTIs) and abortions in sows and was formerly assigned to a variety of genera, including Corynebacterium, Eubacterium, and Actinomyces (9, 17). A. schaalii is a new species recovered from human blood and urine and is suspected to cause UTIs (9, 12). Two newly described species, Actinobaculum massiliae and Actinobaculum urinale, were recovered from the urine of elderly women with chronic cystitis (6, 7). A. massiliae is also described as a new cause of superficial skin infections (16). Problems with identifying Actinobaculum spp. with traditional phenotypic tests have obscured their pathological role for many years. We describe 10 cases of A. schaalii infections.
Nine strains of A. schaalii were available for growth, biochemical, and susceptibility tests. The strains were cultured on nine different culture media at 35°C in ambient air, in air with 5% CO2, and anaerobically. The media were 5% Columbia sheep blood agar (Becton Dickinson [BD], Heidelberg, Germany), 5% and 10% horse blood agar (Statens Serum Institut [SSI], Copenhagen, Denmark), chocolate agar (SSI), Brucella blood agar with hemin and vitamin K1 (BD), anaerobic plates (SSI), nutrient agar plates (SSI), semisolid agar containing pepsin blood and thioglycolate (SSI), and serum broth (SSI). The CAMP reaction was performed on CAMP plates containing sheep erythrocytes (SSI) with a streak of a beta-hemolytic strain of Staphylococcus aureus. The strains were characterized by using the API Coryne and Rapid ID32A systems in accordance with the manufacturer's instructions (API bioMerieux, Marcy l'Etoile, France). Carbohydrate fermentation reactions were read after 24 and 48 h of incubation.
A Quantitect SYBR green kit (QIAGEN) was used with real-time PCR mixtures (50-μl total volume) containing 1x PCR buffer and a 200 μM concentration of each primer. The primers used for amplification of the 16S rRNA gene, BSF-8 (5'-AGAGTTTGATCCTGGCTCAG-3') and BSR-534 (5'-ATTACCGCGGCTGCTGGC-3'), produced a 526-bp fragment of the 16S rRNA gene. Samples of 1 and 5 μl were tested by PCR. PCRs were performed by using an Opticon DNA engine (MJ Research). The amplification profile included incubation at 95°C for 15 min followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR samples were spin column purified using Microcon YM-100 filter units (Millipore) for DNA sequencing. DNA strands of the amplicons were sequenced on an ABI PRISM 3100 Avant genetic analyzer (Applied Biosystems) using BSF-8 and BSR-534 as sequencing primers and a BigDye v.3.1 kit (Applied Biosystems). Sequencing data were edited using SeqScape software (Applied Biosystems), and the data were then compared to deposited sequences in the NCBI database using the BLAST search engine.
MICs for benzylpenicillin, cefuroxime, amdinocillin, nitrofurantoin, ciprofloxacin, tetracycline, gentamicin, and clindamycin were determined with the E-test (AB Biodisk, Solna, Sweden). An inoculum suspension of 1 McFarland standard in 0.9% NaCl was applied to Brucella blood agar containing hemin and vitamin K1 (BD). The MICs were read after 48 h of anaerobic incubation at 35°C. Bacteroides fragilis ATCC 25285 was used as a quality control strain.
Clinical data and predisposing conditions are summarized in Table 1. Patients 1, 3, 5, 6, and 8 had a history of recurrent symptoms of UTI and unexplained pyuria for months or years before A. schaalii was identified.
Our nine isolates were nonmotile, non-acid-fast, non-spore-forming, gram-positive coccoid rods. The isolates grew on all nine media after anaerobic incubation for 3 days, with the largest colonies seen on agar plates containing horse and sheep blood. On 5% Columbia sheep blood agar in an anaerobic atmosphere at 35°C for 48 h, A. schaalii cells grew as gray colonies of <1 mm in diameter. The colonies showed weak -hemolysis on agar plates containing horse and sheep blood after 2 to 5 days. The CAMP reaction was absent. The isolates grew either well or poorly in air with 5% CO2 and either poorly or not at all in ambient air. All strains were catalase and oxidase negative. Our isolates were compared with related species, and general information about the isolates is given in Table 2, while the results with the API Coryne system are given in Table 3. In the manufacturer's database, the API Coryne numerical profiles for the isolates were identified as doubtful or unacceptable profiles for Arcanobacterium bernardiae, Arcanobacterium hemolyticum, Arcanobacterium pyogenes, or Gardenerella vaginalis. Automatic reading of the Rapid ID32A test strip yielded the numerical codes 0400077705 (five strains), 0420077705 (three strains), and 0430077705 (one strain). According to the manufacturer's database, these were identified as very good (0400077705) or unacceptable (0420077705 and 0430077705) profiles for Actinomyces meyeri.
Sequence similarities of 96 to 100% were found by 16S rRNA gene sequencing for all strains matching closely to A. schaalii. The best taxon match and second best taxon match had a major identity score difference. Actinomyces sp. (n = 4), Arcanobacterium sp. (n = 3), Mycobacterium sp. (n = 2), and Myceligeneris sp. (n = 1) were recognized as the phylogenetically closest taxa.
The strains showed only small interisolate susceptibility variations and were susceptible to penicillin, cefuroxime, amdinocillin, nitrofurantoin, tetracycline, and clindamycin, with low MICs (Table 4). Reduced activities were seen with ciprofloxacin and gentamicin. Preliminary E-tests showed in vitro resistance to trimethoprim and sulfamethoxazole.
Our observations support, as recently reported (9, 12), the hypothesis that A. schaalii can cause UTIs in predisposed individuals. The difficulties in isolating and identifying Actinobaculum spp. are known (6, 7, 12). A. schaalii can be overlooked or interpreted as a contaminant due to its slow growth under aerobic conditions and its resemblance to the normal bacterial flora on skin and mucosa.
During 2004, seven strains of A. schaalii were identified in Viborg, Denmark, which has a population of 230,000, by the Department of Clinical Microbiology at Viborg Hospital. In this department, urine cultures are routinely incubated in a CO2-enriched atmosphere. This practice probably facilitates the identification of A. schaalii, and this finding strongly suggests that A. schaalii infections are more common than was previously recognized.
It is recommended that the identification of A. schaalii be done by performing both the API Coryne and Rapid ID32A test systems, at least until the manufacturers' databases have been updated. In doubtful cases, the strains should be referred to a reference laboratory for definite confirmation by 16S rRNA gene sequencing.
In the case of patient 8, treatment failure was observed after therapy with amoxicillin (500 mg three times daily) for 1 week. Since treatment failure with amoxicillin was also reported for a patient with a chronic UTI due to A. massiliae (6), treatment with -lactam antibiotics for a prolonged period may be required.
Microbiologists and clinicians should be aware of A. schaalii and related species in cases of unexplained chronic pyuria, especially if the microscopic findings differ from the growth results under aerobic conditions. In these cases, urine samples should be cultured on appropriate media and incubated in an anaerobic atmosphere.
ACKNOWLEDGMENTS
We thank Karen Marie Sby, Anne-Marie Hesselberg, and Jonna Jensen for their help with finding and describing the strains and Prem Bajaj of Microbiology Department, Viborg Hospital, Denmark, for reviewing the article.
REFERENCES
Almuzara, M. N., C. de Mier, C. M. Barberis, J. Mattera, A. Famiglietti, and C. Vay. 2002. Arcanobacterium hemolyticum: identification and susceptibility to nine antimicrobial agents. Clin. Microbiol. Infect. 8:828-829.
Catlin, B. W. 1992. Gardnerella vaginalis—characteristics, clinical considerations, and controversies. Clin. Microbiol. Rev. 5:213-237.
Funke, G., C. P. Ramos, J. F. Fernandez-Garayzabal, N. Weiss, and M. D. Collins. 1995. Description of human-derived Centers for Disease Control coryneform group 2 bacteria as Actinomyces bernardiae sp. nov. Int. J. Syst. Bacteriol. 45:57-60.
Funke, G., F. N. R. Renaud, J. Freney, and P. Riegel. 1997. Multicenter evaluation of the updated and extended API (RAPID) Coryne database 2.0. J. Clin. Microbiol. 35:3122-3126.
Gahrn-Hansen, B., and W. Frederiksen. 1992. Human infections with Actinomyces pyogenes (Corynebacterium pyogenes). Diagn. Microbiol. Infect. Dis. 15:349-354.
Greub, G., and D. Raoult. 2002. "Actinobaculum massiliae," a new species causing chronic urinary tract infection. J. Clin. Microbiol. 40:3938-3941.
Hall, V., M. D. Collins, R. A. Hutson, E. Falsen, E. Inganas, and B. I. Duerden. 2003. Actinobaculum urinale sp. nov., from human urine. Int. J. Syst. Evol. Microbiol. 53:679-682.
Hall, V., M. D. Collins, P. A. Lawson, R. A. Hutson, E. Falsen, E. Inganas, and B. Duerden. 2003. Characterization of some Actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen. nov., sp. nov. J. Clin. Microbiol. 41:640-644.
Lawson, P. A., E. Falsen, E. Akervall, P. Vandamme, and M. D. Collins. 1997. Characterization of some Actinomyces-like isolates from human clinical specimens: reclassification of Actinomyces suis (Soltys and Spratling) as Actinobaculum suis comb. nov. and description of Actinobaculum schaalii sp. nov. Int. J. Syst. Bacteriol. 47:899-903.
Lepargneur, J. P., R. Heller, R. Soulie, and P. Riegel. 1998. Urinary tract infection due to Arcanobacterium bernardiae in a patient with a urinary tract diversion. Eur. J. Clin. Microbiol. Infect. Dis. 17:399-401.
Mackenzie, A., L. A. Fuite, F. T. H. Chan, J. King, U. Allen, N. Macdonald, and F. Diazmitoma. 1995. Incidence and pathogenicity of Arcanobacterium haemolyticum during a 2-year study in Ottawa. Clin. Infect. Dis. 21:177-181.
Pajkrt, D., A. M. Simoons-Smit, P. H. M. Savelkoul, J. van den Hoek, W. W. M. Hack, and A. M. van Furth. 2003. Pyelonephritis caused by Actinobaculum schaalii in a child with pyeloureteral junction obstruction. Eur. J. Clin. Microbiol. Infect. Dis. 22:438-440.
Ramos, C. P., G. Foster, and M. D. Collins. 1997. Phylogenetic analysis of the genus Actinomyces based on 16S rRNA gene sequences: description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov., and Arcanobacterium pyogenes comb. nov. Int. J. Syst. Bacteriol. 47:46-53.
Sabbe, L. J. M., D. Van de Merwe, L. Schouls, A. Bergmans, M. Vaneechoutte, and P. Vandamme. 1999. Clinical spectrum of infections due to the newly described Actinomyces species A. turicensis, A. radingae, and A. europaeus. J. Clin. Microbiol. 37:8-13.
Sarkonen, N., E. Kononen, P. Summanen, M. Kononen, and H. Jousimies-Somer. 2001. Phenotypic identification of Actinomyces and related species isolated from human sources. J. Clin. Microbiol. 39:3955-3961.
Waghorn, D. J. 2004. Actinobaculum massiliae: a new cause of superficial skin infection. J. Infect. 48:276-277.
Woldemeskel, M., W. Drommer, and M. Wendt. 2002. Microscopic and ultrastructural lesions of the ureter and renal pelvis in sows with regard to Actinobaculum suis infection. J. Vet. Med. Ser. A 49:348-352.(Mark Reinhard, Jrgen Prag)
Department of Clinical Microbiology, Odense Hospital, Odense
Unit of Clinical Microbiology, Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen
Departments of Medicine
Surgery, Roskilde Hospital, Roskilde, Denmark
ABSTRACT
Nine of 10 strains of Actinobaculum schaalii caused urinary tract infections in predisposed individuals. Identification included 16S rRNA gene sequence analysis and use of the API Coryne and Rapid ID32A test systems. A. schaalii is easily overlooked due to its slow growth in ambient air and its resemblance to the normal bacterial flora on skin and mucosa.
TEXT
The genus Actinobaculum, first described in 1997, includes the Actinobaculum suis and Actinobaculum schaalii species. A. suis is an important cause of urinary tract infections (UTIs) and abortions in sows and was formerly assigned to a variety of genera, including Corynebacterium, Eubacterium, and Actinomyces (9, 17). A. schaalii is a new species recovered from human blood and urine and is suspected to cause UTIs (9, 12). Two newly described species, Actinobaculum massiliae and Actinobaculum urinale, were recovered from the urine of elderly women with chronic cystitis (6, 7). A. massiliae is also described as a new cause of superficial skin infections (16). Problems with identifying Actinobaculum spp. with traditional phenotypic tests have obscured their pathological role for many years. We describe 10 cases of A. schaalii infections.
Nine strains of A. schaalii were available for growth, biochemical, and susceptibility tests. The strains were cultured on nine different culture media at 35°C in ambient air, in air with 5% CO2, and anaerobically. The media were 5% Columbia sheep blood agar (Becton Dickinson [BD], Heidelberg, Germany), 5% and 10% horse blood agar (Statens Serum Institut [SSI], Copenhagen, Denmark), chocolate agar (SSI), Brucella blood agar with hemin and vitamin K1 (BD), anaerobic plates (SSI), nutrient agar plates (SSI), semisolid agar containing pepsin blood and thioglycolate (SSI), and serum broth (SSI). The CAMP reaction was performed on CAMP plates containing sheep erythrocytes (SSI) with a streak of a beta-hemolytic strain of Staphylococcus aureus. The strains were characterized by using the API Coryne and Rapid ID32A systems in accordance with the manufacturer's instructions (API bioMerieux, Marcy l'Etoile, France). Carbohydrate fermentation reactions were read after 24 and 48 h of incubation.
A Quantitect SYBR green kit (QIAGEN) was used with real-time PCR mixtures (50-μl total volume) containing 1x PCR buffer and a 200 μM concentration of each primer. The primers used for amplification of the 16S rRNA gene, BSF-8 (5'-AGAGTTTGATCCTGGCTCAG-3') and BSR-534 (5'-ATTACCGCGGCTGCTGGC-3'), produced a 526-bp fragment of the 16S rRNA gene. Samples of 1 and 5 μl were tested by PCR. PCRs were performed by using an Opticon DNA engine (MJ Research). The amplification profile included incubation at 95°C for 15 min followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR samples were spin column purified using Microcon YM-100 filter units (Millipore) for DNA sequencing. DNA strands of the amplicons were sequenced on an ABI PRISM 3100 Avant genetic analyzer (Applied Biosystems) using BSF-8 and BSR-534 as sequencing primers and a BigDye v.3.1 kit (Applied Biosystems). Sequencing data were edited using SeqScape software (Applied Biosystems), and the data were then compared to deposited sequences in the NCBI database using the BLAST search engine.
MICs for benzylpenicillin, cefuroxime, amdinocillin, nitrofurantoin, ciprofloxacin, tetracycline, gentamicin, and clindamycin were determined with the E-test (AB Biodisk, Solna, Sweden). An inoculum suspension of 1 McFarland standard in 0.9% NaCl was applied to Brucella blood agar containing hemin and vitamin K1 (BD). The MICs were read after 48 h of anaerobic incubation at 35°C. Bacteroides fragilis ATCC 25285 was used as a quality control strain.
Clinical data and predisposing conditions are summarized in Table 1. Patients 1, 3, 5, 6, and 8 had a history of recurrent symptoms of UTI and unexplained pyuria for months or years before A. schaalii was identified.
Our nine isolates were nonmotile, non-acid-fast, non-spore-forming, gram-positive coccoid rods. The isolates grew on all nine media after anaerobic incubation for 3 days, with the largest colonies seen on agar plates containing horse and sheep blood. On 5% Columbia sheep blood agar in an anaerobic atmosphere at 35°C for 48 h, A. schaalii cells grew as gray colonies of <1 mm in diameter. The colonies showed weak -hemolysis on agar plates containing horse and sheep blood after 2 to 5 days. The CAMP reaction was absent. The isolates grew either well or poorly in air with 5% CO2 and either poorly or not at all in ambient air. All strains were catalase and oxidase negative. Our isolates were compared with related species, and general information about the isolates is given in Table 2, while the results with the API Coryne system are given in Table 3. In the manufacturer's database, the API Coryne numerical profiles for the isolates were identified as doubtful or unacceptable profiles for Arcanobacterium bernardiae, Arcanobacterium hemolyticum, Arcanobacterium pyogenes, or Gardenerella vaginalis. Automatic reading of the Rapid ID32A test strip yielded the numerical codes 0400077705 (five strains), 0420077705 (three strains), and 0430077705 (one strain). According to the manufacturer's database, these were identified as very good (0400077705) or unacceptable (0420077705 and 0430077705) profiles for Actinomyces meyeri.
Sequence similarities of 96 to 100% were found by 16S rRNA gene sequencing for all strains matching closely to A. schaalii. The best taxon match and second best taxon match had a major identity score difference. Actinomyces sp. (n = 4), Arcanobacterium sp. (n = 3), Mycobacterium sp. (n = 2), and Myceligeneris sp. (n = 1) were recognized as the phylogenetically closest taxa.
The strains showed only small interisolate susceptibility variations and were susceptible to penicillin, cefuroxime, amdinocillin, nitrofurantoin, tetracycline, and clindamycin, with low MICs (Table 4). Reduced activities were seen with ciprofloxacin and gentamicin. Preliminary E-tests showed in vitro resistance to trimethoprim and sulfamethoxazole.
Our observations support, as recently reported (9, 12), the hypothesis that A. schaalii can cause UTIs in predisposed individuals. The difficulties in isolating and identifying Actinobaculum spp. are known (6, 7, 12). A. schaalii can be overlooked or interpreted as a contaminant due to its slow growth under aerobic conditions and its resemblance to the normal bacterial flora on skin and mucosa.
During 2004, seven strains of A. schaalii were identified in Viborg, Denmark, which has a population of 230,000, by the Department of Clinical Microbiology at Viborg Hospital. In this department, urine cultures are routinely incubated in a CO2-enriched atmosphere. This practice probably facilitates the identification of A. schaalii, and this finding strongly suggests that A. schaalii infections are more common than was previously recognized.
It is recommended that the identification of A. schaalii be done by performing both the API Coryne and Rapid ID32A test systems, at least until the manufacturers' databases have been updated. In doubtful cases, the strains should be referred to a reference laboratory for definite confirmation by 16S rRNA gene sequencing.
In the case of patient 8, treatment failure was observed after therapy with amoxicillin (500 mg three times daily) for 1 week. Since treatment failure with amoxicillin was also reported for a patient with a chronic UTI due to A. massiliae (6), treatment with -lactam antibiotics for a prolonged period may be required.
Microbiologists and clinicians should be aware of A. schaalii and related species in cases of unexplained chronic pyuria, especially if the microscopic findings differ from the growth results under aerobic conditions. In these cases, urine samples should be cultured on appropriate media and incubated in an anaerobic atmosphere.
ACKNOWLEDGMENTS
We thank Karen Marie Sby, Anne-Marie Hesselberg, and Jonna Jensen for their help with finding and describing the strains and Prem Bajaj of Microbiology Department, Viborg Hospital, Denmark, for reviewing the article.
REFERENCES
Almuzara, M. N., C. de Mier, C. M. Barberis, J. Mattera, A. Famiglietti, and C. Vay. 2002. Arcanobacterium hemolyticum: identification and susceptibility to nine antimicrobial agents. Clin. Microbiol. Infect. 8:828-829.
Catlin, B. W. 1992. Gardnerella vaginalis—characteristics, clinical considerations, and controversies. Clin. Microbiol. Rev. 5:213-237.
Funke, G., C. P. Ramos, J. F. Fernandez-Garayzabal, N. Weiss, and M. D. Collins. 1995. Description of human-derived Centers for Disease Control coryneform group 2 bacteria as Actinomyces bernardiae sp. nov. Int. J. Syst. Bacteriol. 45:57-60.
Funke, G., F. N. R. Renaud, J. Freney, and P. Riegel. 1997. Multicenter evaluation of the updated and extended API (RAPID) Coryne database 2.0. J. Clin. Microbiol. 35:3122-3126.
Gahrn-Hansen, B., and W. Frederiksen. 1992. Human infections with Actinomyces pyogenes (Corynebacterium pyogenes). Diagn. Microbiol. Infect. Dis. 15:349-354.
Greub, G., and D. Raoult. 2002. "Actinobaculum massiliae," a new species causing chronic urinary tract infection. J. Clin. Microbiol. 40:3938-3941.
Hall, V., M. D. Collins, R. A. Hutson, E. Falsen, E. Inganas, and B. I. Duerden. 2003. Actinobaculum urinale sp. nov., from human urine. Int. J. Syst. Evol. Microbiol. 53:679-682.
Hall, V., M. D. Collins, P. A. Lawson, R. A. Hutson, E. Falsen, E. Inganas, and B. Duerden. 2003. Characterization of some Actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen. nov., sp. nov. J. Clin. Microbiol. 41:640-644.
Lawson, P. A., E. Falsen, E. Akervall, P. Vandamme, and M. D. Collins. 1997. Characterization of some Actinomyces-like isolates from human clinical specimens: reclassification of Actinomyces suis (Soltys and Spratling) as Actinobaculum suis comb. nov. and description of Actinobaculum schaalii sp. nov. Int. J. Syst. Bacteriol. 47:899-903.
Lepargneur, J. P., R. Heller, R. Soulie, and P. Riegel. 1998. Urinary tract infection due to Arcanobacterium bernardiae in a patient with a urinary tract diversion. Eur. J. Clin. Microbiol. Infect. Dis. 17:399-401.
Mackenzie, A., L. A. Fuite, F. T. H. Chan, J. King, U. Allen, N. Macdonald, and F. Diazmitoma. 1995. Incidence and pathogenicity of Arcanobacterium haemolyticum during a 2-year study in Ottawa. Clin. Infect. Dis. 21:177-181.
Pajkrt, D., A. M. Simoons-Smit, P. H. M. Savelkoul, J. van den Hoek, W. W. M. Hack, and A. M. van Furth. 2003. Pyelonephritis caused by Actinobaculum schaalii in a child with pyeloureteral junction obstruction. Eur. J. Clin. Microbiol. Infect. Dis. 22:438-440.
Ramos, C. P., G. Foster, and M. D. Collins. 1997. Phylogenetic analysis of the genus Actinomyces based on 16S rRNA gene sequences: description of Arcanobacterium phocae sp. nov., Arcanobacterium bernardiae comb. nov., and Arcanobacterium pyogenes comb. nov. Int. J. Syst. Bacteriol. 47:46-53.
Sabbe, L. J. M., D. Van de Merwe, L. Schouls, A. Bergmans, M. Vaneechoutte, and P. Vandamme. 1999. Clinical spectrum of infections due to the newly described Actinomyces species A. turicensis, A. radingae, and A. europaeus. J. Clin. Microbiol. 37:8-13.
Sarkonen, N., E. Kononen, P. Summanen, M. Kononen, and H. Jousimies-Somer. 2001. Phenotypic identification of Actinomyces and related species isolated from human sources. J. Clin. Microbiol. 39:3955-3961.
Waghorn, D. J. 2004. Actinobaculum massiliae: a new cause of superficial skin infection. J. Infect. 48:276-277.
Woldemeskel, M., W. Drommer, and M. Wendt. 2002. Microscopic and ultrastructural lesions of the ureter and renal pelvis in sows with regard to Actinobaculum suis infection. J. Vet. Med. Ser. A 49:348-352.(Mark Reinhard, Jrgen Prag)